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背景:神经元异常放电是癫痫的基本特征,这种异常放电的基础是细胞内外离子的跨膜运动,然而癫痫时是否存在钙离子内流,神经元膜上的钙通道是否开放还不清楚。目的:了解钙离子及钙通道在癫痫发病中的作用,探讨马桑毒内酯(coriamyrtiry,CM)调节神经细胞内钙稳态的机制。设计:培养的原代神经元随机分成:正常对照组;致痫组;尼莫地平组。利用激光扫描共聚焦显微镜技术观察胞内游离钙离子浓度的变化。地点和对象:以培养的Wistar乳鼠海马锥体神经元为靶细胞进行测试,实验在四川大学华西医院完成。干预:正常对照组不加任何条件;致痫组加入不同浓度的致痫剂CM。根据加入CM浓度的不同,又分为致痫10mL/L组、致痫20mL/L组、致痫40mL/L组、致痫80mL/L组。尼莫地平组同时加入CM(40mL/L)和尼莫地平。三组均培养24h后以Fluo-3进行负载,利用激光扫描共聚焦显微镜检测。主要观察指标:测量并记录锥体神经元和背景的荧光强度,以相对荧光强度代表游离钙离子值。结果:正常组神经细胞内Ca2+浓度为95.43±10.52,经致痫剂CM作用后,神经细胞内Ca2+浓度致痫10mL/L组为1299.87±68.53,致痫20mL/L组为1333.00±55.99,致痫40mL/L组为1315.33±55.73,致痫80mL/L组为1379.47±57.11,高于正常组(P<0.01),但其升高不呈剂量依赖性(P>0.05);经
BACKGROUND: Abnormal discharge of neurons is the basic characteristic of epilepsy. The basis of this abnormal discharge is the transmembrane movement of ions inside and outside the cells. However, it is still unclear whether there is influx of calcium ions in epilepsy and whether the calcium channels in neuronal membranes are open. Objective: To investigate the role of calcium and calcium channels in the pathogenesis of epilepsy and to explore the mechanism by which coriamyrtiry (CM) regulates calcium homeostasis in nerve cells. Design: Primary cultured neurons were randomly divided into: normal control group; epileptogenic group; nimodipine group. Laser scanning confocal microscopy was used to observe the change of intracellular free calcium concentration. Location and Subjects: The cultured hippocampal pyramidal neurons of Wistar suckling mice were used as target cells. Experiments were performed at West China Hospital of Sichuan University. Intervention: normal control group without any condition; epileptic group added different concentrations of epileptogenic agent CM. According to the different concentration of CM, it was divided into epilepsy 10 mL / L group, epilepsy 20 mL / L group, epilepsy 40 mL / L group and epilepsy 80 mL / L group. The nimodipine group was added with CM (40 mL / L) and nimodipine simultaneously. All of the three groups were cultured with Fluo-3 for 24 hours and then detected by laser scanning confocal microscopy. MAIN OUTCOME MEASURES: The fluorescence intensity of pyramidal neurons and background was measured and recorded, and the relative fluorescence intensity represented the value of free calcium. Results: The Ca2 + concentration of neurons in normal group was 95.43 ± 10.52. After CM induced by epileptogenic agent, the concentration of Ca2 + in intracellular Ca2 + group was 1299.87 ± 68.53, while that in 20mL / L group was 1333.00 ± 55.99 (P <0.01), but the dose-dependent manner was not significant (P> 0.05). After the treatment of epilepsy, the incidence of epilepsy was 1315.33 ± 55.73 in the 40 mL / L group and 1379.47 ± 57.11 in the 80 mL / L epilepsy group